The regulation of dimethyl fumarate in the differentiation of Th17 cells in vitro
Objective:To investigate the inhibitory effect of dimethyl fumarate(DMF)on the differentiation of helper T cells 17(Th17)in vitro.Methods:Lymphocytes were obtained from the systemic lymph nodes and spleens of C57BL/6J mice.Naïve CD4+T cells were sorted by immunomagnetic beads and induced to differentiate into Th17 cells under the action of cytokines,and the induction differenti-ation model of Th17 cells was established in vitro.The experiment was divided into control group,5 μmol/L DMF group,15 μmol/L DMF group and 25 μmol/L DMF group.The ratio of Th17 cells in each group was detected by flow cytometry.The relative expression of retinoic acid receptor-related orphan receptor gamma-t(RORγt),interleukin-17A(IL-17A),interleukin-23 receptor(IL-23R),granulocyte-macrophage colony-stimulating factor(GM-CSF),interferon gamma(IFN-γ),T-box expressed in T cells(T-bet)and forkhead box protein P3(FOXP3)mRNA in each group was detected by qRT-PCR.The protein expression of IL-17A in each group was detected by ELISA.Results:The positive sorting rate of CD4+T cells was about 93%.The results of flow cytometry showed that compared with the control group,the ratio of Th17 cells was significantly decreased after DMF treatment,and the inhibitory effect of 25 μmol/L DMF was the most significant(t=5.227,P<0.01)in a dose-dependent manner.There was no effect on cell activity at 25 μmol/L DMF concentration.The results of qRT-PCR analysis showed that the relative mRNA expression of RORγt,IL-17A,IL-23R,GM-CSF and IFN-γ in the 25 μmol/L DMF group were significantly lower than those in the control group,and the difference was statistically significant(t=4.061,P<0.05;t=4.701,P<0.01;t=19.18,P<0.0001;t=19.18,P<0.05;t=2.870,P<0.05,respectively),but there was no significant difference in the expression of T-bet and FOXP3 mRNA between the two groups(t=0.105、0.7319,both P＞0.05).The results of ELISA showed that the concentration of IL-17 A in the 25 μmol/L DMF group was significantly lower than that in the control group(t=4.786,P<0.01).Conclusion:DMF may inhibit the differentiation of Naïve CD4+T cells into Th17 cells and the pathogenic function of Th17 cells by inhibiting the expression of RORγt,IL-17A,IL-23R,GM-CSF and IFN-γ.
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